Flow cytometry, the measurement of cells in a moving liquid stream, is a valuable analysis tool in research laboratories. Conventional flow cytometry devices for sorting objects such as cells and particles basically consist of a liquid stream forming a sheath fluid into which cell sample is introduced then focused through an orifice. As the objects pass through the orifice, particular characteristics of the objects are determined based upon the analyzing or counting capabilities of the device. Usually, the device can sort or count at high speeds, collecting tens of thousands of the objects based on a variety of chemical and physical characteristics such as size, granulation of the cytoplasm and presentation of specific antigens. Accordingly, there has been considerable interest in flow cytometry to sort objects for subsequent analysis.
One commercially available flow cytometer which relies on a hydrodynamically focused fluid system is known as the FACScan TM instrument sold by Becton Dickinson Immunocytometry Systems, San Jose, Calif. The FACScan TM instrument rapidly analyzes cells on the basis of fluorescence and light scatter properties. Analysis is accomplished by introducing cells in a suspension to the center of a focused liquid stream thus causing them to pass, one at a time, through a focused light from a high powered laser. Each cell is individually characterized by its light scatter signals and by the intensity and color of fluorescence emitted while it is illuminated. This system is described in U.S. Pat. No. 4,844,610 issued Jul. 4, 1989 to North, U.S. Pat. No. 5,030,022 issued Jul. 9, 1991 to North and U.S. Pat. No. 5,040,890 issued Aug. 20, 1991 to North.
Typically flow cytometers systems have been implemented as pressure driven fluidics systems driven by pressure pumps. However, pressure driven systems have proven disadvantageous in that system leaks produce sprays of sheath fluid which may expose the operator to bio-hazardous substances and cause damage to optical and electronic components of the instrument. Regulatory valves required to control pressure driven cytometry systems tend to become clogged with blood cells causing the valves to stick or otherwise malfunction. In addition, the design of pressure driven fluidics systems is more complicated than is the design of vacuum driven fluidics systems since pressure driven systems require the use of pressurized connections for the supply reservoir and for other features necessary for the system to withstand high system pressure levels. Pressure driven systems also require the sample vessel to sealably engage the flow cell assembly. Removal of the sample vessel can produce hazardous aerosols and back flow dripping of bio-hazardous fluids.
Thus, a vacuum driven flow cytometry system provides many advantages over a pressure driven system. The design of the supply reservoir is greatly simplified, not requiring the use of pressurizing connections, and can be refilled by gravity drain from a elevated supply vessel. In addition, there is no back-flow drip from the cell sample uptake tube, used to introduce the cell sample into the sheath fluid flow, which may expose the operator to bio-hazardous substances. Cell sample vessels do not have to be pressurized or sealably engage the instrument to contain the system fluid allowing a new freedom of design in terms of the size and shape of the cell sample vessel. This freedom of design facilitates the design of auxiliary equipment, such as automatic tube lifters, which improve cell sample presentation. Another advantage realized is that the descent of the tube lifter can be prolonged allowing fluid residue from the sample uptake tube to drain into the cell sample vessel thus minimizing cell sample carryover which could skew test results of subsequent test runs. Finally, use of vacuum driven fluidics provides the opportunity to design a system in which the pump is utilized on a "demand" basis, being turned off once the system has reached a predetermined system vacuum or pressure level thus increasing the service life of the pump.
A major problem encountered in the development of a vacuum driven flow cytometer is the creation of air bubbles in the sheath fluid as the system pressure level is reduced to a level below atmospheric pressure. Air dissolved in the sheath fluid at atmospheric pressure comes out of solution when the sheath fluid is subjected to a vacuum. The bubbles lodge in troublesome areas such as in the analysis region of the flow cell. The bubbles may deflect cells from their proper trajectory through the illuminated area or analysis region of the flow cell. The present invention solves this problem through the use of a deaerator connected to the intake passage of the flow cell which removes much of the air dissolved in the sheath fluid.
Another problem presented in the development of a vacuum driven flow cytometer is air being drawn into the flow cell by the residual vacuum remaining in the flow cell at the end of the test cycle. Thus, a means is required to equalize the vacuum developed in the flow cell thereby preventing air from being drawn into the flow cell. This problem is solved by providing a valve, connected to an outlet passage of the flow cell, which vents the flow cell to atmospheric pressure as the tube lifter is descending thereby preventing air from being drawn into the flow cell. Computer control of valve actuation and the rate of descent of the tube lifter allows synchronous programmed control of the timing of these components. A rate of tube lifter descent which is sufficiently slow to permit the flow cell to fully vent before the uptake tube is removed from the analysis region is specified in programmed control.